Claims for Patent: 4,663,281

Summary for Patent: 4,663,281

Title:	Enhanced production of proteinaceous materials in eucaryotic cells
Abstract:	Disclosed is a method of enhancing expression of recombinant DNA in eucaryotic cells. A tissue specific enhancer element obtained from the genome of an organism and active in a selected tissue type is combined with a transcriptionally competent transcription unit comprising a promoter and exons encoding for the proteinaceous material of interest (or its precursor). This recombinant DNA is transfected into cells derived from the same tissue as the tissue in which the enhancer element normally functions to enhance expression of endogeneous DNA. The resulting transformants express the exons of the transcription unit at high levels as the enhancer element increases the copy number of mRNA. The enhancer element operates to increase transcription independent of its orientation and position provided it is located within an active region on the DNA, generally between about 1-10 kilobases (kb) from the 3\' or 5\' end of the transcription unit.
Inventor(s):	Gillies; Stephen D. (Scituate, MA), Tonegawa; Susumu (Chestnut Hill, MA)
Assignee:	Mass Institute of Technology (Cambridge, MA)
Application Number:	06/592,231
Patent Claims:	1. A process for producing a proteinaceous material in a mammalian cell line derived from a selected tissue type comprising the steps of: combining DNA comprising a mammalian a tissue specific cellular enhancer element with DNA comprising a transcription unit encoding said proteinaceous material or a precursor thereof to produce transcriptionally competent recombinant DNA, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operable naturally to increase the production of an endogenous proteinaceous substance; transfecting cells of said mammalian cell line with said recombinant DNA; and culturing said transfected cell line to produce enhanced quantities of said proteinaceous material. 2. The process of claim 1 wherein said enhancer element, when present in the endogenous genome of a cell from said selected tissue type, functions to enhance production of a proteinaceous substance selected from the group consisting of albumin, globulins, fibrinogen, and hormones. 3. The process of claim 1 wherein said combining step includes combining said recombinant DNA with a vector selected from the group consisting of plasmids and viruses. 4. The process of claim 3 wherein said vector further includes expressable DNA which corresponds to a gene coding for a selectable marker. 5. The process of claim 1 wherein said transfecting and culturing steps result in a stably transformed cell line. 6. The process of claim 1 wherein said enhancer element, when present in the endogenous genome of a lymphoid cell, functions to enhance expression of DNA encoding a globulin. 7. The process of claim 1 wherein said enhancer element comprises at least a portion of the nucleotide sequences set forth in FIG. 10 and said cell line is a lymphoid cell line. 8. The process of claim 1 wherein said cell line is a myeloma cell line and said enhancer element, when present in the endogenous genome of a lymphoid cell, enhances the production of an immunoglobulin. 9. The process of claim 1 wherein the cell line transfected with said recombinant DNA is derived from the same cell type as the cell in which said enhancer element is active. 10. A vector for transfecting a mammalian cell derived from a selected tissue type to produce a cell line which secretes a proteinaceous material, said vector comprising an exon encoding said proteinaceous material or a precursor thereof and a promoter sequence, and recombined therewith, tissue specific mammalian cellular enhancer element at a site within an active region of said vector sufficiently close to said transcription unit to enhance production of mRNA independent of its orientation and position within said active region, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operable naturally to increase production of an endogenous proteinaceous substance. 11. The vector of claim 10 wherein said enhancer element, when present in the endogenous genome of a cell from said selected tissue type, functions to enhance the production of a proteinaceous substance selected from the group consisting of albumin, globulins, fibrinogen, and hormones. 12. The vector of claim 10 further comprising expressable DNA which corresponds to a gene coding for a selectable marker. 13. The vector of claim 10 comprising a member selected from the group consisting of plasmids and viruses. 14. The vector of claim 10 wherein said enhancer element and said transcription unit are derived from different cell lines. 15. The vector of claim 10 wherein said enhancer element, when present in the endogenous genome of a lymphoid cell, is operative naturally to enhance production of immunoglobulin. 16. The vector of claim 10 wherein said enhancer element, when present as part of the endogenous genome of a lymophoid cell in an intron of a .gamma.2b heavy chain gene, serves to enhance production of a .gamma.2b heavy chain protein. 17. The vector of claim 10 wherein said enhancer element comprises at least a portion of the nucleotide sequences set forth in FIG. 10. 18. A mammalian cell transformant for producing a proteinaceous material, said transformant comprising a genetically modified cell derived from a selected mammalian tissue type containing a transfected DNA comprising: a transcription unit comprising an exon encoding said proteinaceous material or a precursor thereof and a promoter sequence; and, recombined therewith, tissue specific a mammalian cellular enhancer element at a site within an active region of said DNA sufficiently close to said transcription unit to enhance production of mRNA independent of orientation and position within said active region, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operative naturally to enhance production of an endogenous proteinaceous substance. 19. The transformant of claim 18 wherein said enhancer element, when present in the endogenous genome of a cell from said selected tissue type, functions to enhance the production of a proteinaceous substance selected from the group consisting of albumin, globulins, fibrinogen, and hormones. 20. The transformant of claim 18 wherein said genetically modified cell is a lymphoid cell and said enhancer element, when present in the endogenous genome of a lymphoid cell, enhances the production of an immunoglobulin. 21. The transformant of claim 18 comprising a stably transformed cell. 22. The transformant of claim 20 wherein said enhancer element comprises at least a portion of the nucleotide sequences set forth in FIG. 10. 23. The transformant of claim 18 comprising a genetically modified cell. 24. A vector for receiving DNA encoding a proteinaceous material and for transfecting a mammalian cell derived from a selected tissue type to produce a cell line which produces said proteinaceous material, said vector comprising a restriction site for receiving a gene encoding said proteinaceous material and tissue specific a mammalian cellular enhancer element located within an active region of said vector sufficiently close to said restriction site to enhance production of mRNA corresponding to a gene inserted into said restriction site, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operable naturally to increase production of an endogenous proteinaceous substance. 25. The vector of claim 27 further comprising a promoter upstream of said restriction site.

Details for Patent 4,663,281

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26